JPET # 256594 R2 Effect of Adenylyl Cyclase Type 6 …jpet.aspetjournals.org/content/jpet/early/2019/05/08/...JPET # 256594 R2 1 Effect of Adenylyl Cyclase Type 6 on Localized Production

JPET # 256594 R2

1

Effect of Adenylyl Cyclase Type 6 on Localized Production of cAMP by Beta-2

Adrenoceptors in Human Airway Smooth Muscle Cells

1Shailesh R. Agarwal, 1Chase Fiore, 1Kathryn Miyashiro,

2Rennolds S. Ostrom, and 1Robert D. Harvey

1Department of Pharmacology,

University of Nevada, Reno School of Medicine, Reno NV 89557

and

2Department of Biomedical and Pharmaceutical Sciences,

Chapman University School of Pharmacy, Irvine, CA 92618

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 8, 2019 as DOI: 10.1124/jpet.119.256594

at ASPE

T Journals on February 12, 2020

jpet.aspetjournals.orgD

ownloaded from

http://jpet.aspetjournals.org/

JPET # 256594 R2

2

Running Title: cAMP Compartmentation in Airway Smooth Muscle

Address for Correspondence: Robert D. Harvey

Department of Pharmacology

University of Nevada, Reno

1664 N. Virginia Street, MS 573

Reno, NV 89557

Tel 775-784-4119

Fax 775-784-1620

Email: [email protected]

Text pages: 31

Tables: 0

Figures: 6

References: 31

Abstract words: 249

Introduction words: 748

Discussion words: 868


at ASPE



ownloaded from


JPET # 256594 R2

3

Abbreviations:

AC6, adenylyl cyclase type 6; AKAP, A kinase-anchoring protein; ASM, airway smooth

muscle; β2AR, β2-adrenoceptor; ECFP, enhanced cyan fluorescent protein; EP2R, EP2

prostaglandin receptor; Epac2-camps, exchange protein activated by cAMP type 2-

based cAMP biosensor; Epac2-CAAX, Epac2-camps biosensor with a prenylation

targeting sequence; Epac2-MyrPalm, Epac2-camps biosensor with an acylation

targeting sequence; EYFP, enhanced yellow fluorescent protein; FRET, fluorescence

resonance energy transfer; Fsk, forskolin; IBMX, 3-isobutyl-1-methylxanthine; Iso,

isoproterenol; PDE4, phosphodiesterase type 4; PKA, protein kinase A

Recommended Section Assignment: Cellular and Molecular


at ASPE



ownloaded from


JPET # 256594 R2

4

Abstract

β2-Adrenoceptors (β2ARs) are concentrated in caveolar lipid raft domains of the plasma

membrane in airway smooth muscle (ASM) cells, along with adenylyl cyclase type 6

(AC6). This is believed to contribute to how these receptors can selectively regulate

certain types of cAMP-dependent responses in these cells. The goal of the present

study was to test the hypothesis that β2AR production of cAMP is localized to specific

subcellular compartments using fluorescence resonance energy transfer (FRET)-based

cAMP biosensors targeted to different microdomains in human ASM cells. Epac2-

MyrPalm and Epac2-CAAX biosensors were used to measure responses associated

with lipid raft and non-raft regions of the plasma membrane, respectively. Activation of

β2ARs with isoproterenol produced cAMP responses most readily detected in lipid raft

domains. Furthermore, overexpression of AC6 somewhat paradoxically inhibited β2AR

production of cAMP in lipid raft domains, without affecting β2AR responses detected in

other subcellular locations or cAMP responses to EP2 prostaglandin receptor activation,

which were confined primarily to non-raft domains of the plasma membrane. The

inhibitory effect of overexpressing AC6 was blocked by inhibition of phosphodiesterase

type 4 (PDE4) activity with rolipram, inhibition of protein kinase A (PKA) activity with

H89, and inhibition of A kinase anchoring protein (AKAP) interactions with the peptide

inhibitor Ht31. These results support the idea that overexpression of AC6 leads to

enhanced feedback activation of PDE4 via phosphorylation by PKA that is part of an

AKAP-dependent signaling complex. This provides insight into the molecular basis for

localized regulation of cAMP signaling in human ASM cells.


at ASPE



ownloaded from


JPET # 256594 R2

5

Introduction

β2-adrenoceptor (β2AR) agonists are a mainstay of therapy for asthma, chronic

obstructive pulmonary disease and other respiratory disorders. β2AR activation induces

airway smooth muscle (ASM) relaxation and bronchodilation by stimulating adenylyl

cyclase (AC) activity and the production of cAMP. While cAMP plays a central role in

regulating the contractile state of ASM (Pelaia et al., 2008; Noble et al., 2014), this

ubiquitous signaling molecule is also involved in eliciting a number of other responses in

the muscle cells that make up this tissue (Billington et al., 2013). The specific effect

mediated by cAMP often depends on the signaling pathway responsible for its

production. In ASM cells, cAMP production is regulated by both β2ARs and EP2

prostaglandin receptors (EP2Rs) (Bogard et al., 2011). Production of cAMP by either

type of receptor is capable of causing human ASM relaxation. However, only cAMP

produced by β2ARs stimulates cytoskeletal reorganization and cell shape changes

associated with arborization and only cAMP produced by EP2Rs increases the

expression of the cytokine IL-6 (Bogard et al., 2012; Bogard et al., 2014).

Understanding how cells compartmentalize cAMP and generate unique responses upon

activation of a given receptor is important, because it may help explain why β2AR

agonists, which are widely used as bronchodilators, suffer from issues with

tachyphylaxis due to receptor desensitization (Penn et al., 1998) and increased adverse

effects (Taylor, 2009).

The simplest way to explain the ability of different receptors to illicit unique functional

responses is if each receptor stimulates cAMP production that is confined to distinct


at ASPE



ownloaded from


JPET # 256594 R2

6

subcellular locations. However, for this to be true, these receptors must then be

distributed in distinctly different physical locations. Consistent with this hypothesis,

β2ARs and EP2Rs have been found to reside in different fractions of the plasma

membrane (Ostrom et al., 2001; Bogard et al., 2011; Bogard et al., 2012). Most cell

membranes contain cholesterol- and sphingomyelin-rich domains referred to as lipid

rafts, which form buoyant membrane fractions on a sucrose density gradient (Insel et

al., 2005; Ostrom and Insel, 2006). Caveolae are represented by a subset of the

buoyant fractions associated specifically with caveolins, which are scaffolding proteins

involved in forming signaling complexes. In human ASM cells, β2ARs are found

specifically in caveolar, lipid raft fractions of the plasma membrane, while EP2Rs are

found in the non-raft membrane fractions (Bogard et al., 2011).

ASM responses to β2AR and EP2R stimulation have also been attributed to activation of

different isoforms of adenylyl cyclase (AC), the enzyme responsible for cAMP

production. AC type 6 (AC6) is found specifically in caveolar membrane fractions of

ASM cells, and its overexpression selectively enhances β2AR production of cAMP and

cAMP-dependent arborization. AC type 2 (AC2) is found exclusively in non-raft fractions

of the plasma membrane, and its overexpression selectively enhances EP2R stimulation

of cAMP and cAMP-dependent production of IL-6 (Bogard et al., 2011; Bogard et al.,

2012; Bogard et al., 2014).

The purpose of the present study was to test the hypothesis that in primary human ASM

cells β2AR stimulation preferentially enhances cAMP production in subcellular locations


at ASPE



ownloaded from


JPET # 256594 R2

7

associated with lipid raft domains of the plasma membrane by a mechanism involving

activation of AC6. This was done by comparing cAMP responses detected by Epac2-

camps, a FRET-based biosensor, expressed in different subcellular locations (Nikolaev

et al., 2004). Epac2-camps itself has no targeting sequences and is expressed

throughout the cytoplasmic domain. Epac2-MyrPalm is a version of the probe that

contains an acylation sequence targeting it to lipid raft domains of the plasma

membrane, and Epac2-CAAX contains a prenylation sequence that targets the probe to

non-raft domains of the plasma membrane. These sequences have been previously

shown to target proteins to lipid raft and non-raft fractions of the plasma membrane of

various cell types (Melkonian et al., 1999; Zacharias et al., 2002; Depry et al., 2011).

Furthermore, our previous work has also demonstrated that probes targeted using these

sequences are capable of detecting differences in responses produced by receptors

located in lipid raft and non-raft membrane domains of various cell types (Agarwal et al.,

2014; Agarwal et al., 2017; Agarwal et al., 2018). In human ASM cells, we

demonstrated the EP2Rs preferentially couple to AC2 to produce changes in cAMP that

are confined primarily to non-raft domains of the plasma membrane (Agarwal et al.,

2017). In the present study, we demonstrate that β2ARs produce responses that are

primarily associated with lipid raft domains. Furthermore, AC6 regulates this response

through a mechanism that involves direct interaction with protein kinase A (PKA) and

positive feedback activation of PDE4.


at ASPE



ownloaded from


JPET # 256594 R2

8

Materials and Methods

Cell Culture. Human ASM cells were derived from tracheae and primary bronchi from

non-asthmatic human donors by Dr. Raymond Penn (Thomas Jefferson University) and

Dr. Reynold Panettieri (Rutgers University), as described previously (Yan et al., 2011).

These cells come from de-identified biological samples that are considered exempt by

the University of Nevada, Reno and Chapman University Institutional Review Boards.

Experiments were conducted using multiple samples of primary cells derived from six

different patients representing both sexes. All cells used were between passage 6 and

9. Cells were maintained in Ham’s F12 nutrient mixture supplemented with 10% fetal

bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin. Cells were transduced

with adenovirus constructs containing Epac2-based cAMP biosensors for 48-72 hours.

For overexpression experiments, adenoviral constructs expressing AC6 and/or Ht31

were used at titers previously shown to produce >90% transduction efficiency (Bogard

et al., 2011). All experiment were conducted at room temperature.

FRET Microscopy. The Epac2-camps, Epac2-MyrPalm, and Epac2-CAAX

biosensors used in the present study have been previously characterized (Nikolaev et

al., 2004; Agarwal et al., 2014; Agarwal et al., 2017; Agarwal et al., 2018). All three

probes have a similar affinity for activation by cAMP (Agarwal et al., 2014). Live cell

imaging experiments were conducted as described previously (Warrier et al., 2005;

Warrier et al., 2007; Iancu et al., 2008; Agarwal et al., 2017). Briefly, cells were bathed

in the following solution (in mM): NaCl 137, KCl 5.4, MgCl2 0.5, CaCl2 1.0, NaH2PO4

0.33, glucose 5.5, and HEPES 5 (pH 7.4). FRET imaging was conducted using an

Olympus IX71 inverted microscope (Olympus America, Center Valley, PA, USA)


at ASPE



ownloaded from


JPET # 256594 R2

9

equipped with a Hamamatsu OrcaD2 dual chip CCD camera and HCImage data

acquisition and analysis software (Hamamatsu Corporation, Bridgewater, NJ, USA).

Images were obtained with a 40x water- immersion objective (1.3 numerical aperture).

ECFP excitation was achieved by using a 300-W xenon arc lamp with a D436/20 band-

pass filter and a 455DCLP dichroic mirror. ECFP and EYFP (FRET) emissions were

measured simultaneously using a 505DCXR dichroic mirror with D480/30 and D535/30

band-pass filters. Changes in cAMP activity were defined as the change in background

and bleed-through corrected ECFP/EYFP fluorescence intensity ratio (ΔR) relative to

the baseline ratio (R0) measured in a region of interest. As long as the nucleus was

avoided, moving the ROI did not significantly affect the results. Ratiometric

measurements also ensured that the responses measured were independent of the

level of expression of the probes. FRET ratios were measured once every 10 seconds.

A baseline of at least 5 min was established prior to the application of any drug.

Because the response to low concentrations of agonists were relatively slow to develop,

all drugs were applied for at least 10 min before measuring their effects. Responses

were calculated relative to the average ratio measured over the 30 s period immediately

preceding application of any drug. To control for any differences in the dynamic range of

the FRET responses produced by the different probes, all results were normalized to

the magnitude of the maximal probe response observed in the same cell following

exposure to maximally stimulating concentrations 3-isobutlyl-1-methylxanthine (IBMX),

a non-specific PDE inhibitor, plus forskolin, a direct activator of AC activity.

Materials. Ham’s F12 medium, penicillin, streptomycin, and fetal bovine serum

were purchased from Life Technologies (Carlsbad, CA, USA). Rolipram and forskolin


at ASPE



ownloaded from


JPET # 256594 R2

10

were obtained from Tocris Bioscience (Bristol, UK). All other reagents were purchased

from Sigma-Aldrich (St. Louis, MO, USA).

Statistics. All data are expressed as the mean ± SEM of the indicated number of

experiments conducted using different cells (n) obtained from different preparations (N).

Statistical significance (P < 0.05) was determined by Student’s t-test with Holm-Sidak

correction, where appropriate, using SigmaPlot (Systat Software, San Jose, CA, USA).


at ASPE



ownloaded from


JPET # 256594 R2

11

Results

Responses to β2AR stimulation were measured using submaximally stimulating

concentrations of the agonist isoproterenol (Iso). In control cells, exposure to 1 nM Iso

elicited responses that were detected by the cytosolic (Epac2-camps) and lipid raft

(Epac2-MyrPalm) targeted probes, but not by the non-raft targeted (Epac2-CAAX)

probe (figure 1A). Exposure to 3 nM Iso elicited responses detected by all three probes,

although the response detected by Epac2-CAAX was significantly smaller (figure 1C).

These results are consistent with the idea that β2ARs are primarily concentrated in lipid

raft domains of the plasma membrane of human ASM cells.

We then examined the effect of overexpressing adenylyl cyclase type 6 (AC6). This did

not affect the responses to maximally stimulating concentrations of forskolin plus IBMX

(see supplement figure 1). However, it did significantly reduce the relative magnitude of

the response to β2AR stimulation detected by Epac2-MyrPalm (P < 0.05), without

altering the responses detected by Epac2-camps or Epac2-CAAX (P > 0.05) (figure 1B,

C). This supports the idea that AC6 is targeted primarily to lipid raft domains of the

plasma membrane, where it affects β2AR-mediated production of cAMP. Despite the

fact that AC6 overexpression selectively altered β2AR responses associated with lipid

raft domains, our original hypothesis was that it would enhance the production of cAMP.

Contrary to this prediction, AC6 overexpression actually inhibited the β2AR response

detected by Epac2-MyrPalm.


at ASPE



ownloaded from


JPET # 256594 R2

12

To determine if the effect of overexpressing AC6 is specific to β2ARs, we also looked at

the effect it had on responses to EP2 prostaglandin receptor (EP2R) activation using the

agonist butaprost. As reported previously, in control cells, butaprost elicited cAMP

responses that were most readily detected by the Epac2-CAAX probe in non-raft

domains of the plasma membrane (figure 2A) (Agarwal et al., 2017). Furthermore,

overexpressing AC6 neither enhanced nor inhibited cAMP responses to butaprost

(P > 0.05) (figure 2B, C) detected by any of the probes. This further supports the idea

that AC6 is selectively associated with regulating β2AR responses in lipid raft domains

of the plasma membrane.

We hypothesized that AC6 overexpression causes a localized decrease in cAMP

produced by β2ARs in lipid raft domains as a result of an increase in phosphodiesterase

(PDE) activity through a positive feedback mechanism involving protein kinase A (PKA)

(Conti and Beavo, 2007). The activity of PDE4, which is prominent in airway smooth

muscle cells (Méhats et al., 2003; Bogard et al., 2012), has been shown to be

upregulated by PKA-dependent phosphorylation. To examine this possibility, we looked

at the effect of rolipram (10 µM), a selective PDE4 inhibitor, on β2AR responses in

control cells as well as cells overexpressing AC6 (figure 3). Rolipram by itself had no

effect on cAMP activity detected by the Epac2-MyrPalm probe in either control or AC6

overexpressing cells (P > 0.05). Furthermore, rolipram did not appear to alter the

magnitude of the response to Iso in control cells (figure 3A). However, it did significantly

alter the response to Iso in AC6 overexpressing cells (figure 3B, C). In the presence of

rolipram, the response to Iso was not significantly different from control cells (P > 0.05).


at ASPE



ownloaded from


JPET # 256594 R2

13

This is in contrast to the significant decrease in magnitude of the β2AR response

associated with AC6 overexpression in the absence of PDE4 inhibition (see figure 1).

Furthermore, this effect appeared to be specific for responses associated with lipid raft

domains, as rolipram did not enhance the response to Iso detected by Epac2-CAAX in

non-raft domains of AC6 overexpressing cells (figure 3B, C), it was the same size as the

response to Iso in control cells in the absence of rolipram (see figure 1A, C). This

supports the idea that AC6 overexpression leads to a localized decrease in cAMP

produced by β2ARs associated specifically with lipid raft domains that is mediated by

PDE4.

The above results are consistent with the hypothesis that overexpressing AC6 leads to

an increase in cAMP production, which in turn acts in a positive feedback manner to

enhance PDE4 activity, presumably through a mechanism involving PKA-dependent

phosphorylation (Conti and Beavo, 2007). To examine this possibility, we compared the

response to Iso in cells first exposed to the PKA inhibitor H89 (1 µM), to prevent the

feedback activation of PDE4. The PKA inhibitor itself did not produce a change in basal

cAMP activity (figure 4). However, H89 did appear to alter the Iso-stimulated cAMP

response detected by Epac2-MyrPalm in AC6 overexpressing cells (figure 4B). In the

presence of H89, the response to 3 nM Iso was not significantly different from control

cells (P > 0.05) (figure 4C). Again, this is contrast to the significant decrease in

magnitude of the β2AR response associated with AC6 overexpression in the absence of

H89 (see figure 1B, C). This effect appeared to be specific for responses associated


at ASPE



ownloaded from


JPET # 256594 R2

14

with lipid raft domains, as H89 did not enhance the β2AR responses detected in non-raft

domains of AC6 overexpressing cells (figure 4C).

The above results support the idea that positive feedback activation of PDE4

contributes to the decrease in cAMP produced in response to β2AR stimulation in lipid

raft domains of the plasma membrane, but only in AC6 overexpressing cells. This could

be explained if overexpression of AC6 leads to recruitment of excess PKA activity.

Adenylyl cyclases have been shown to be integral components of signaling complexes

associated with type II PKA through interactions with A kinase anchoring proteins

(AKAPs) (Dessauer, 2009). To determine if this type of interaction contributes to the

effect of AC6 overexpression, we compared responses to Iso in cells overexpressing

the Ht31 peptide, which disrupts PKA anchoring to AKAPs (Carr et al., 1992). We found

that in the presence of the Ht31 peptide, the response to 3 nM Iso detected by Epac2-

MyrPalm in cells overexpressing AC6 was no different in magnitude than the response

observed in control cells (P > 0.05) (figure 5A, B). This suggests that the decrease in

magnitude of the Iso response detected by Epac2-MyrPalm in AC6 overexpressing cells

in the absence of Ht31 (see figure 1B, C) involves the recruitment of PKA activity to lipid

raft domains where β2AR signaling occurs. Again, this effect appears to be specific for

responses associated with lipid raft domains, as Ht31 did not alter the Iso response

detected by Epac2-CAAX (P > 0.05) (figure 5A, B).


at ASPE



ownloaded from


JPET # 256594 R2

15

Discussion

In the present study, we found that activation of β2ARs with Iso caused an increase in

cAMP activity that could be detected throughout the cytoplasmic domain of human ASM

cells, as well subcellular locations associated with lipid raft and non-raft domains of the

plasma membrane. However, changes associated with lipid raft domains were more

sensitive to β2AR stimulation and larger than those associated with non-raft domains.

Furthermore, overexpression of AC6 selectively altered β2AR production of cAMP in

lipid raft domains, without affecting cAMP responses occurring in other locations or

cAMP responses mediated by other receptors. In addition, the effect that AC6

overexpression had on β2AR responses in lipid raft domains was inhibitory, and this

inhibitory effect could be blocked by inhibition of PDE4 activity, PKA kinase activity, as

well as AKAP interactions with PKA.

The ability of AC6 overexpression to selectively alter β2AR responses associated with

lipid raft domains is consistent with previous findings demonstrating that both signaling

proteins are concentrated in caveolar fractions of the plasma membrane in ASMs

(Bogard et al., 2011; Bogard et al., 2012). However, the fact that the AC6

overexpression produced an inhibitory effect in the present study was somewhat

surprising. It was previously reported that overexpression of AC6 enhances cAMP

production in response to direct activation of AC activity with forskolin or β2ARs with Iso.

This apparent contradiction is most likely explained by the fact that the earlier studies

measured cAMP activity in cell lysates using traditional immunoassay techniques that

required the use of maximally stimulating concentrations of the non-specific PDE


at ASPE



ownloaded from


JPET # 256594 R2

16

inhibitor IBMX. The results of the present study suggest that the effects of AC6

overexpression are more subtle, in that they only appear to occur in specific subcellular

locations and they can be blocked by selective inhibition of PDE4 activity. Therefore, the

use of whole cell lysates and IBMX would have obscured the ability to detect the kind of

response found presently, using targeted biosensors in intact cells.

The ability to block the effect of AC6 overexpression on β2AR responses with rolipram

as well as the PKA kinase inhibitor H89 supports the idea that the inhibitory effect was

due to an increase in PDE4 activity. It is well documented that PDE4 activity can be

stimulated following phosphorylation by PKA (Conti and Beavo, 2007). It is conceivable

that the increased expression of AC activity contributed to an initial increase in cAMP

production upon β2AR stimulation that resulted in a rapid activation of PKA and

subsequent phosphorylation of PDE4. If this was the case, one might have expected to

see a rapid increase followed by a decrease in cAMP following exposure to Iso. One

possibility is that it occurred too rapidly for us to detect, especially since we were only

able to measure FRET responses once every 10 seconds. It could also be that the

increase in cAMP needed to activate PKA occurred locally, within a signaling complex

that may not have been accessible to the Epac2-MyrPalm biosensor, even though they

are all associated with the same membrane domain.

Consistent with the idea that the β2AR and AC6 are part of a signaling complex that

includes PKA is the fact that the inhibitory effect of AC6 overexpression was also

blocked by the AKAP inhibitory peptide Ht31. This peptide mimics the AKAP anchoring


at ASPE



ownloaded from


JPET # 256594 R2

17

sequence that binds to the regulatory subunit of type II PKA. Preventing the anchoring

of PKA would have limited the ability of local activation of PKA to phosphorylate PDE4.

The implication is the PDE4 is also part of this signaling complex. Although we do not

know which specific AKAP is involved, several different isoforms have been found to

interact with certain AC subtypes and can regulate compartmentalized cAMP signaling

(Horvat et al., 2012). For example, AKAP79/AKAP150/AKAP5 is able to interact with

AC2, 3, 5, 6, 8, and 9 (Efendiev et al., 2010); yotiao/AKAP9 has been shown to interact

with AC1, 2, 3, and 9 (Piggott et al., 2008); and mAKAPβ/AKAP6 is able to interact with

AC2 and 5 (Kapiloff et al., 2009). Although the evidence presented in the current study

supports the idea that AKAP anchoring of PKA is necessary to see the effect of AC6

overexpression, it is possible that the signaling complex, which appears to include

β2ARs, AC6, PDE4, and PKA (figure 6), may involve other scaffolding proteins such as

caveolins (Ostrom et al., 2012). In either case, inhibition of PDE4 activity might have

been expected to affect β2AR responses detected by Epac2-MyrPalm in non-AC6

overexpressing cells (see figure 3A). One possible reason it did not is that other PDE

isoforms are present that can compensate for the loss of PDE4 activity. Only when

overexpression of AC6 recruits excess PDE4 activity to that microdomain is an effect

apparent.

In conclusion, using targeted FRET-based biosensors, we were able to demonstrate

that β2AR stimulation produces a compartmentalized cAMP response associated with

lipid raft domains of the plasma membrane in human ASM cells. Furthermore,

overexpression of AC6 activity revealed functional evidence that the β2AR is associated


at ASPE



ownloaded from


JPET # 256594 R2

18

with a signaling complex that includes AC6, PKA, and PDE4. These findings imply that

therapeutic strategies involving combined β2AR activation and PDE4 inhibition might be

particularly effective at increasing cAMP levels and bronchodilation in human ASM.


at ASPE



ownloaded from


JPET # 256594 R2

19

Acknowledgements

The authors thank Dr. Raymond Penn and Dr. Tonio Pera, Thomas Jefferson

University, for supplying the human ASM cells.


at ASPE



ownloaded from


JPET # 256594 R2

20

Authorship Contributions

Participated in research design: Ostrom and Harvey

Conducted experiments: Agarwal, Fiore, and Miyashiro

Contributed new reagents or analytic tools: Harvey and Agarwal

Performed analysis: Agarwal, Fiore, and Miyashiro

Wrote or contributed to the writing of the manuscript: Agarwal, Ostrom, and Harvey


at ASPE



ownloaded from


JPET # 256594 R2

21

References

Agarwal SR, Gratwohl J, Cozad M, Yang PC, Clancy CE and Harvey RD (2018)

Compartmentalized cAMP Signaling Associated With Lipid Raft and Non-raft Membrane

Domains in Adult Ventricular Myocytes. Front Pharmacol 9:332.

Agarwal SR, Miyashiro K, Latt H, Ostrom RS and Harvey RD (2017)

Compartmentalized cAMP responses to prostaglandin EP2 receptor activation in human

airway smooth muscle cells. Br J Pharmacol 174:2784-2796.

Agarwal SR, Yang PC, Rice M, Singer CA, Nikolaev VO, Lohse MJ, Clancy CE and

Harvey RD (2014) Role of membrane microdomains in compartmentation of cAMP

signaling. PLoS One 9:e95835.

Billington CK, Ojo OO, Penn RB and Ito S (2013) cAMP regulation of airway smooth

muscle function. Pulm Pharmacol Ther 26:112-120.

Bogard AS, Adris P and Ostrom RS (2012) Adenylyl cyclase 2 selectively couples to E

prostanoid type 2 receptors, whereas adenylyl cyclase 3 is not receptor-regulated in

airway smooth muscle. J Pharmacol Exp Ther 342:586-595.

Bogard AS, Birg AV and Ostrom RS (2014) Non-raft adenylyl cyclase 2 defines a cAMP

signaling compartment that selectively regulates IL-6 expression in airway smooth

muscle cells: differential regulation of gene expression by AC isoforms. Naunyn

Schmiedebergs Arch Pharmacol 387:329-339.


at ASPE



ownloaded from


JPET # 256594 R2

22

Bogard AS, Xu C and Ostrom RS (2011) Human bronchial smooth muscle cells express

adenylyl cyclase isoforms 2 , 4 , and 6 in distinct membrane microdomains. J

Pharmacol Exp Ther 337:209-217.

Carr DW, Hausken ZE, Fraser ID, Stofko-Hahn RE and Scott JD (1992) Association of

the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein.

Cloning and characterization of the RII-binding domain. J Biol Chem 267:13376-13382.

Conti M and Beavo J (2007) Biochemistry and physiology of cyclic nucleotide

phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev

Biochem 76:481-511.

Depry C, Allen MD and Zhang J (2011) Visualization of PKA activity in plasma

membrane microdomains. Mol Biosyst 7:52-58.

Dessauer CW (2009) Adenylyl cyclase--A-kinase anchoring protein complexes: the next

dimension in cAMP signaling. Mol Pharmacol 76:935-941.

Efendiev R, Samelson BK, Nguyen BT, Phatarpekar PV, Baameur F, Scott JD and

Dessauer CW (2010) AKAP79 interacts with multiple adenylyl cyclase (AC) isoforms

and scaffolds AC5 and -6 to alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate

(AMPA) receptors. J Biol Chem 285:14450-14458.


at ASPE



ownloaded from


JPET # 256594 R2

23

Horvat SJ, Deshpande DA, Yan H, Panettieri RA, Codina J, DuBose TD, Jr., Xin W,

Rich TC and Penn RB (2012) A-kinase anchoring proteins regulate compartmentalized

cAMP signaling in airway smooth muscle. FASEB J 26:3670-3679.

Iancu RV, Ramamurthy G, Warrier S, Nikolaev VO, Lohse MJ, Jones SW and Harvey

RD (2008) Cytoplasmic cAMP concentrations in intact cardiac myocytes. Am J Physiol

Cell Physiol 295:C414-C422.

Insel PA, Head BP, Ostrom RS, Patel HH, Swaney JS, Tang CM and Roth DM (2005)

Caveolae and lipid rafts: G protein-coupled receptor signaling microdomains in cardiac

myocytes. Ann N Y Acad Sci 1047:166-172.

Kapiloff MS, Piggott LA, Sadana R, Li J, Heredia LA, Henson E, Efendiev R and

Dessauer CW (2009) An adenylyl cyclase-mAKAPbeta signaling complex regulates

cAMP levels in cardiac myocytes. J Biol Chem 284:23540-23546.

Méhats C, Jin SLCL, Wahlstrom J, Law E, Umetsu DT and Conti M (2003) PDE4D

plays a critical role in the control of airway smooth muscle contraction. FASEB J

17:1831-1841.

Melkonian KA, Ostermeyer AG, Chen JZ, Roth MG and Brown DA (1999) Role of lipid

modifications in targeting proteins to detergent-resistant membrane rafts. Many raft

proteins are acylated, while few are prenylated. J Biol Chem 274:3910-3917.


at ASPE



ownloaded from


JPET # 256594 R2

24

Nikolaev VO, Bunemann M, Hein L, Hannawacker A and Lohse MJ (2004) Novel single

chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279:37215-

37218.

Noble PB, Pascoe CD, Lan B, Ito S, Kistemaker L, Tatler AL, Pera T, Brook BS, Gosens

R and West AR (2014) Airway smooth muscle in asthma: Linking contraction and

mechanotransduction to disease pathogenesis and remodelling. Pulm Pharmacol Ther

29:96-107.

Ostrom RS, Bogard AS, Gros R and Feldman RD (2012) Choreographing the adenylyl

cyclase signalosome: sorting out the partners and the steps. Naunyn Schmiedebergs

Arch Pharmacol 385:5-12.

Ostrom RS, Gregorian C, Drenan RM, Xiang Y, Regan JW and Insel PA (2001)

Receptor number and caveolar co-localization determine receptor coupling efficiency to

adenylyl cyclase. J Biol Chem 276:42063-42069.

Ostrom RS and Insel PA (2006) Methods for the study of signaling molecules in

membrane lipid rafts and caveolae. Methods Mol Biol 332:181-191.

Pelaia G, Renda T, Gallelli L, Vatrella A, Busceti M, Agati S, Caputi M, Cazzola M,

Maselli R and Marsico SA (2008) Molecular mechanisms underlying airway smooth

muscle contraction and proliferation: Implications for asthma. Respir Med 102:1173-

1181.


at ASPE



ownloaded from


JPET # 256594 R2

25

Penn RB, Panettieri RA, Jr. and Benovic JL (1998) Mechanisms of acute

desensitization of the beta2AR-adenylyl cyclase pathway in human airway smooth

muscle. Am J Respir Cell Mol Biol 19:338-348.

Piggott LA, Bauman AL, Scott JD and Dessauer CW (2008) The A-kinase anchoring

protein Yotiao binds and regulates adenylyl cyclase in brain. Proc Natl Acad Sci USA

105:13835-13840.

Taylor DR (2009) The beta-agonist saga and its clinical relevance: on and on it goes.

Am J Respir Crit Care Med 179:976-978.

Warrier S, Belevych AE, Ruse M, Eckert RL, Zaccolo M, Pozzan T and Harvey RD

(2005) Beta-adrenergic and muscarinic receptor induced changes in cAMP activity in

adult cardiac myocytes detected using a FRET based biosensor. Am J Physiol Cell

Physiol 289:C455-C461.

Warrier S, Ramamurthy G, Eckert RL, Nikolaev VO, Lohse MJ and Harvey RD (2007)

cAMP microdomains and L-type Ca2+ channel regulation in guinea-pig ventricular

myocytes. J Physiol (Lond) 580:765-776.

Yan H, Deshpande DA, Misior AM, Miles MC, Saxena H, Riemer EC, Pascual RM,

Panettieri RA and Penn RB (2011) Anti-mitogenic effects of beta-agonists and PGE2 on

airway smooth muscle are PKA dependent. FASEB J 25:389-397.


at ASPE



ownloaded from


JPET # 256594 R2

26

Zacharias DA, Violin JD, Newton AC and Tsien RY (2002) Partitioning of lipid-modified

monomeric GFPs into membrane microdomains of live cells. Science 296:913-916.


at ASPE



ownloaded from


JPET # 256594 R2

27

Footnotes

This work was supported by the National Institutes of Health [Grants R01 GM107094,

R01 GM101928, and R01 HL145778]. SRA was supported by a National Institute of

General Medical Sciences Center of Biological Research Excellence [P20 GM130459].


at ASPE



ownloaded from


JPET # 256594 R2

28

Figure Legends

Figure 1 Changes in cAMP activity in different subcellular domains following β2AR

stimulation. Representative time-course of changes in magnitude of FRET-response

(ΔR/R0) detected by Epac2-MyrPalm, Epac2-CAAX, and Epac2-camps biosensors

following activation of β2ARs with 1 nM and 3 nM isoproterenol (Iso) in (A) untreated

human airway smooth muscle (ASM) cells (control) and (B) human ASM cells

overexpressing adenylyl cyclase type 6 (AC6 OE). Results are normalized to the

magnitude of the maximal response produced by forskolin (FSK, 10 µM) and IBMX (100

µM) in the same cell. (C) Size of average FRET responses in control cells (black bars)

and AC6 OE cells (red bars). AC6 OE caused a significant (P < 0.05, student’s t-test)

decrease in the magnitude of the responses detected by Epac2-MyrPalm (control,

n/N = 6/6; AC6 OE, n/N = 9/4), but not those detected by Epac2-CAAX (control,

n/N = 7/6; AC6 OE, n/N = 6/6), or Epac2-camps (control, n/N = 6/3; AC6 OE, n/N = 5/3).

Figure 2 Changes in cAMP activity in different subcellular domains following EP2R

stimulation. Representative time-course of changes in magnitude of FRET-response

(ΔR/R0) detected by Epac2-MyrPalm, Epac2-CAAX, and Epac2-camps biosensors

following activation of EP2Rs with 0.3 µM and 1 µM butaprost in (A) untreated human

airway smooth muscle (ASM) cells (control) and (B) human ASM cells overexpressing

adenylyl cyclase type 6 (AC6 OE). Results are normalized to the magnitude of the

maximal response produced by forskolin (FSK, 10 µM) and IBMX (100 µM) in the same


at ASPE



ownloaded from


JPET # 256594 R2

29

cell. (C) Size of average FRET responses in control cells (black bars) and AC6 OE cells

(red bars). AC6 OE did not significantly affect (P > 0.05, student’s t-test) responses

detected by Epac2-MyrPalm (control, n/N = 13/8; AC6 OE, n/N = 8/5), Epac2-CAAX

(control, n/N = 20/10; AC6 OE, n/N = 10/7), or Epac2-camps (control, n/8 = 13; AC6 OE,

n/N = 11/6).

Figure 3 Changes in cAMP activity associated with lipid raft and non-raft plasma

membrane domains following β2AR stimulation in the presence of phosphodiesterase

type 4 (PDE4) inhibition. Representative time-course of changes in magnitude of FRET-

response (ΔR/R0) detected by Epac2-MyrPalm and Epac2-CAAX biosensors following

inhibition of PDE4 activity with rolipram (10 µM) and subsequent activation of β2ARs

with 3 nM isoproterenol (Iso) in (A) untreated human airway smooth muscle (ASM)

cells (control) and (B) human ASM cells overexpressing adenylyl cyclase type 6 (AC6

OE). Results are normalized to the magnitude of the maximal response produced by

forskolin (FSK, 10 µM) and IBMX (100 µM) in the same cell. (C) Size of average FRET

responses in control cells (black bars) and AC6 OE cells (red bars). In the presence of

rolipram, there were no significant (P > 0.05, students t-test) differences in the

magnitude of the responses to Iso detected by Epac2-MyrPalm in control (n/N = 6/5)

and AC6 OE (n/N = 6/4) cells or those detected by Epac2-CAAX in control (n/N = 9/5)

and AC6 OE (n/N = 96) cells.


at ASPE



ownloaded from


JPET # 256594 R2

30


membrane domains following β2AR stimulation in the presence of protein kinase A

(PKA) inhibition. Representative time-course of changes in magnitude of FRET-

response (ΔR/R0) detected by Epac2-MyrPalm and Epac2-CAAX biosensors following

inhibition of PKA activity with H89 (1 µM) and subsequent activation of β2ARs with 3 nM

isoproterenol (Iso) in (A) untreated human airway smooth muscle (ASM) cells (control)

and (B) human ASM cells overexpressing adenylyl cyclase type 6 (AC6 OE). Results

are normalized to the magnitude of the maximal response produced by forskolin (FSK,

10 µM) and IBMX (100 µM) in the same cell. (C) Size of average FRET responses in

control cells (black bars) and AC6 OE cells (red bars). In the presence of H89, there

were no significant (P > 0.05, students t-test) differences in the magnitude of the

responses to Iso detected by Epac2-MyrPalm in control (n/N = 6/4) and AC6 OE

(n/N = 6/4) cells or those detected by Epac2-CAAX in control (n/N = 9/7) and AC6 OE

(n/N = 6/4) cells.


membrane domains following β2AR stimulation in cells overexpressing the A kinase

anchoring protein (AKAP) disrupting peptide Ht31. (A) Representative time-course of

changes in magnitude of FRET-response (ΔR/R0) detected by Epac2-MyrPalm and

Epac2-CAAX biosensors following activation of β2ARs with 3 nM isoproterenol (Iso) in

human airway smooth muscle (ASM) cells overexpressing Ht31 and adenylyl cyclase

type 6 (AC6 OE). Results are normalized to the magnitude of the maximal response

produced by forskolin (FSK, 10 µM) and IBMX (100 µM) in the same cell. (B) Size of


at ASPE



ownloaded from


JPET # 256594 R2

31

average FRET responses in control cells (black bars) and cells overexpressing AC6

plus Ht31 (red bars). There were no significant (P > 0.05, students t-test with Holm-

Sidek correction) differences in the magnitude of the responses to Iso detected by

Epac2-MyrPalm in control cells (n/N = 6/6) and cells overexpressing AC6 plus Ht31)

(n/N = 10/4) or the magnitude of the responses detected by Epac2-CAAX in control cells

(n/N = 7/6) and cells overexpressing Ht31 plus AC6 (n/N = 7/3). Control data from

experiments illustrated in figure 1.

Figure 6 Compartmentalized cAMP signaling in human airway smooth muscle cells.

β2AR stimulation of adenylyl cyclase type 6 (AC6) produces cAMP that is most readily

detected by the Epac2-MyrPalm biosensor, which is targeted to lipid raft domains of the

plasma membrane, and the Epac2-camps biosensor, which is expressed throughout the

cytosolic domain. A kinase anchoring proteins (AKAPs) contribute to the formation of a

signaling complex that includes β2ARs, AC6, type II protein kinase A (PKA), and

phosphodiesterase type 4 (PDE4) associated with lipid raft membrane domains. EP2

prostaglandin receptor (EP2Rs) stimulation of AC type 2 (AC2) produces cAMP that is

most readily detected by the Epac2-CAAX biosensor, which is targeted to non-raft

membrane domains.


at ASPE



ownloaded from


Time (min)0 10 20 30 40

0

30

60

90

Time (min)0 10 20 30 40

0

30

60

90

Time (min)0 10 20 30 40

R

/R0

(%

max

)

0

30

60

901 nM Iso

1 nM 3 nM

R/R

0 (

% m

ax)

0

30

60

90

Epac2-campsEpac2-CAAXEpac2-MyrPalm

Time (min)0 10 20 30 40

0

30

60

90

Time (min)0 10 20 30 40

0

30

60

90

Time (min)0 10 20 30 40

R/R

0 (%

max

)

0

30

60

90

1 nM 3 nM0

30

60

90

Figure 1

CONTROL

AC6 OE

3 nM Iso

Fsk+IBMX

1 nM Iso

3 nM Iso

Fsk+IBMX

1 nM3 nM

Fsk+IBMX

1 nM Iso

3 nM Iso

Fsk+IBMX

1 nM Iso

3 nM Iso

Fsk+IBMX

1 nM 3 nM

Fsk+IBMX

1 nM 3 nM0

30

60

90

ns

*ns

ns

ns

*

isoproterenol isoproterenolisoproterenol

A

B

C

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

JPET

Fast Forward. Published on M

ay 8, 2019 as DO

I: 10.1124/jpet.119.256594 at ASPET Journals on February 12, 2020 jpet.aspetjournals.org Downloaded from


nsns nsns

Time (min)0 10 20 30 40

R

/R0

(% m

ax)

0

30

60

90

Time (min)0 10 20 30 40

0

30

60

90

Time (min)0 10 20 30 40

0

30

60

90

0.3

R/R

0 (

% m

ax)

0

30

60

Epac2-campsEpac2-CAAXEpac2-MyrPalm

Time (min)0 10 20 30 40

0

30

60

90

Time (min)0 10 20 30 40

0

30

60

90

Time (min)0 10 20 30 40

R/R

0 (

% m

ax)

0

30

60

90

Figure 2

CONTROL

AC6 OE

Fsk+IBMXFsk+IBMXFsk+IBMX

Fsk+IBMXFsk+IBMXFsk+IBMX

1 M0.3

1 M

0.31 M0.3

1 M

0.31 M

0

30

60

0.31 M

ns

0

30

60

ns

butaprost

0.3 M 1 M

butaprost

0.3 M 1 M

butaprost

0.3 M 1 M

A

B

C




JPET


ay 8, 2019 as DO



ns

ns

ns

ns

Time (min)0 10 20 30 40 50

0

30

60

90

Time (min)0 10 20 30 40

R

/R0

(%

max

)

0

30

60

90

rol

rol rol + Iso0

30

60

90

Epac2-MyrPalm Epac2-CAAX

Time (min)0 10 20 30 40

0

30

60

90

Time (min)0 10 20 30 40 50

R/R

0 (%

max

)

0

30

60

90

rol rol + Iso

R/R

0 (

% m

ax)

0

30

60

90

Figure 3

CONTROL

AC6 OE

Iso

Fsk+IBMX

rol

Iso

Fsk+IBMX

rol

Iso

Fsk+IBMX

rol

Iso

Fsk+IBMX

A

B

C




JPET


ay 8, 2019 as DO



ns

ns

ns

ns

Time (min)0 10 20 30 40

0

30

60

90

Time (min)0 10 20 30 40

R

/R0

(%

max

)

0

30

60

90

H89

H89 H89+Iso

R/R

0 (

% m

ax)

0

20

40

60

Epac2-MyrPalm Epac2-CAAX

Time (min)0 10 20 30 40

0

30

60

90

Time (min)0 10 20 30 40

R/R

0 (

% m

ax)

0

30

60

90

H89 H89+Iso

0

20

40

60

Figure 4

CONTROL

AC6 OE

Iso

Fsk+IBMX

H89

Iso

Fsk+IBMX

H89

Iso

Fsk+IBMX

H89

Iso

Fsk+IBMX

A

B

C




JPET


ay 8, 2019 as DO



Time (min)0 10 20 30

R

/R0

(%

max

)

0

30

60

90

R

/R0

(%

max

)

0

30

60

90

Epac2-CAAXEpac2-MyrPalm

Time (min)0 10 20 30

0

30

60

90

0

30

60

90

Figure 5

AC6 OE

+Ht31

Fsk+IBMXFsk+IBMX

+Ht31

Iso

Iso

ns

ns

control AC6+Ht31

control AC6+Ht31

A

B




JPET


ay 8, 2019 as DO



PDE4

β2AR

Epac2-camps

Epac2-MyrPalm

Epac2-CAAX

EP2R

Gs

AC2

cAMP

AC6

cAMP

AKAP

PKA

Figure 6

Gs




JPET


ay 8, 2019 as DO



Documents

JPET # 256594 R2 Effect of Adenylyl Cyclase Type 6 …jpet.aspetjournals.org/content/jpet/early/2019/05/08/...JPET # 256594 R2 1 Effect of Adenylyl Cyclase Type 6 on Localized Production